The single extracytoplasmic-function sigma factor of Xylella fastidiosa is involved in the heat shock response and presents an unusual regulatory mechanism

Genome sequence analysis of the bacterium Xylella fastidiosa revealed the presence of two genes, named rpoE and rseA, predicted to encode an extracytoplasmic function (ECF) sigma factor and an anti-sigma factor, respectively. In this work, an rpoE null mutant was constructed in the citrus strain J1a12 and shown to be sensitive to exposure to heat shock and ethanol. To identify the X. fastidiosa sigma(E) regulon, global gene expression profiles were obtained by DNA microarray analysis of bacterial cells under heat shock, identifying 21 sigma(E)-dependent genes. These genes encode proteins belonging to different functional categories, such as enzymes involved in protein folding and degradation, signal transduction, and DNA restriction modification and hypothetical proteins. Several putative sigma(E)-dependent promoters were mapped by primer extension, and alignment of the mapped promoters revealed a consensus sequence similar to those of ECF sigma factor promoters of other bacteria. Like other ECF sigma factors, rpoE and rseA were shown to comprise an operon in X. fastidiosa, together with a third open reading frame (XF2241). However, upon heat shock, rpoE expression was not induced, while rseA and XF2241 were highly induced at a newly identified sigma(E)-dependent promoter internal to the operon. Therefore, unlike many other ECF sigma factors, rpoE is not autoregulated but instead positively regulates the gene encoding its putative anti-sigma factor.     

DegU-phosphate activates expression of the anti-sigma factor FlgM in Bacillus subtilis

The bacterial flagellum is a complex molecular machine that is assembled by more than 30 proteins and is rotated to propel cells either through liquids or over solid surfaces. Flagellar gene expression is extensively regulated to co-ordinate flagellar assembly in both space and time. In Bacillus subtilis, the proteins of unknown function, SwrA and SwrB, and the alternative sigma factor σ(D) are required to activate expression of the flagellar filament protein, flagellin. Here we determine that in the absence of SwrA and SwrB, the phosphorylated form of the response regulator DegU inhibits σ(D) -dependent gene expression indirectly by binding to the P(flgM) promoter region and activating expression of the anti-sigma factor FlgM. We further demonstrate that DegU-P-dependent activation of FlgM is essential to inhibit flagellin expression when flagellar basal body assembly is disrupted. Regulation of FlgM is poorly understood outside of Salmonella, and differential control of FlgM expression may be a common means of coupling flagellin expression to flagellar assembly.     

degS (hhoB) is an essential Escherichia coli gene whose indispensable function is to provide σE activity

DegS (HhoB), a putative serine protease related to DegP/HtrA, regulates the basal and induced activity of the essential Escherichia coli sigma factor sigma (E), which is involved in the cellular response to extracytoplasmic stress. DegS promotes the destabilization of the sigma (E)-specific anti-sigma factor RseA, thereby releasing sigma (E) to direct gene expression. We demonstrate that degS is an essential E. coli gene and show that the essential function of DegS is to provide the cell with sigma (E) activity. We also show that the putative active site of DegS is periplasmic and that DegS requires its N-terminal transmembrane domain for its sigma (E)-related function.     

Interactions between the Rhodobacter sphaeroides ECF sigma factor, sigma(E), and its anti-sigma factor, ChrR

Rhodobacter sphaeroides sigma(E) is a member of the extra cytoplasmic function sigma factor (ECF) family, whose members have been shown to regulate gene expression in response to a variety of signals. The functions of ECF family members are commonly regulated by a specific, reversible interaction with a cognate anti-sigma factor. In R.sphaeroides, sigma(E) activity is inhibited by ChrR, a member of a newly discovered family of zinc containing anti-sigma factors. We used gel filtration chromatography to gain insight into the mechanism by which ChrR inhibits sigma(E) activity. We found that formation of the sigma(E):ChrR complex inhibits the ability of sigma(E) to form a stable complex with core RNA polymerase. Since the sigma(E):ChrR complex inhibits the ability of the sigma factor to bind RNA polymerase, we sought to identify amino acid substitutions in sigma(E) that altered the sensitivity of this sigma factor to inhibition by ChrR. This analysis identified single amino acid changes in conserved region 2.1 of sigma(E) that either increased or decreased the sensitivity of sigma(E) for inhibition by ChrR. Many of the amino acid residues that alter the sensitivity of sigma(E) to ChrR are located within regions known to be important for interacting with core RNA polymerase in other members of the sigma(70) superfamily. Our results suggest a model where solvent-exposed residues with region 2.1 of sigma(E) interact with ChrR to sterically occlude this sigma factor from binding core RNA polymerase and to inhibit target gene expression.     

The Bacillus subtilis sigmaW anti-sigma factor RsiW is degraded by intramembrane proteolysis through YluC

The Bacillus subtilis sigma(W) regulon is induced by different stresses such as alkaline shock, salt shock, phage infection and certain antibiotics that affect cell wall biosynthesis. The activity of the alternative, extracytoplasmic function (ECF) sigma factor sigma(W) is modulated by a specific anti-sigma factor (RsiW or YbbM) encoded by the rsiW (ybbM) gene located immediately downstream of sigW. The RsiW membrane topology was determined, and a specific reporter system for RsiW function was constructed. Experiments using the yeast two-hybrid system suggested a direct interaction of sigma(W) with the cytoplasmic part of RsiW. Analysis of truncated forms of the RsiW protein revealed that sigma(W) induction by alkaline shock is dependent on both the transmembrane and the extracytoplasmic domain of RsiW. Western blot and pulse-chase experiments demonstrated degradation of RsiW after an alkaline shock. A B. subtilis mutant strain deleted for the Escherichia coli yaeL orthologue yluC, encoding a transmembrane protease, was defective in inducing a sigma(W)-controlled promoter after alkaline shock and accumulated a membrane-bound truncated form of RsiW, suggesting that the activity of sigma(W) is controlled by the proteolysis of RsiW by at least two different proteolytic steps.